PROJECT SUMMARY/ABSTRACT The human ether-a-go-go gene (hERG) and KCNQ1 genes encode proteins that conduct the cardiac repolarizing currents IKr and IKs, respectively. Impaired repolarization increases the risk of potentially lethal ventricular arrhythmias and is a hallmark of long QT syndrome (LQTS) and heart failure. Treatments for impaired repolarization are limited largely due to their high incidence of off-target effects. hERG cytoplasmic domains interact to negatively modulate IKr, making them potential targets to treat diseases of repolarization. The preliminary data for this grant demonstrate that small antibody peptide fragments targeting distinct regions of the cytoplasmic hERG Per-Arnt-Sim (PAS) domain selectively increase IKr and shorten action potential duration (APD). As a therapeutic, such fragments would potentially shorten APD and restore impaired repolarization in LQTS and heart failure. Additionally, antibody fragments directed toward the PAS and other domains would be useful tools to characterize how specific gating processes regulate cellular behavior and overall cardiac physiology. In the mentored phase of this project, I will characterize the mechanisms by which these antibody fragments modify hERG gating using electrophysiological and spectroscopy techniques. I will also characterize antibody fragment hERG modulation in healthy human stem cell-derived cardiomyocytes (iPSC-CMs) and iPSC-CMs obtained from LQTS patients using scFvs delivered intracellularly via the recording pipette. In the independent phase of this project, I will combine the scFv antibodies with two intracellular delivery techniques: (1) the cell-penetrating Cardiac Targeting Peptide, CTP, and (2) the adeno-associated virus serotype 9 (AAV9). I will introduce antibody-CTP fusion proteins into iPSC-CMs and monitor the resulting biophysical and physiological effects with patch clamp and micro-electrode techniques. To develop an approach leading to a longer-term treatment, I will use the viral AAV9 to produce stable transfer of the antibody-encoding sequence into mammalian cardiomyocytes. These experiments will generate novel tools to probe the mechanistic role of distinct channel processes in native physiology. Additionally, these experiments will act as a proof-of-concept for future experiments designed to develop anti-hERG antibody fragments into treatments for impaired repolarization. This proposal is designed to fulfill my short-term goals of expanding my skills in cardiac electrophysiology and transitioning into the independent phase of my career. This will ultimately allow me to obtain my long-term goal of studying translational cardiac electrophysiology research by using patient-derived iPSC-CMs to explore triggers and treatments for cardiac arrhythmia.